The present invention relates to an accurate and convenient quantitative determination method of mannose by an enzyme method and a reagent for the quantitative determination.
Mannose which is a type of a hexose, exists in human blood in a slight amount. As the route of supply, it has been known that mannose is supplied mainly by a glucose metabolic system, not by ingestion of foods. The concentration of mannose in blood is usually as slight as about 0.5 mg/dl. However, it is known that the concentration tends to be high in the cases of patients suffering from diabetes who lacks the ability of controlling the blood sugar (Clinica. Chimica Acta 251; 1996 pp91-103) or fungal infection (J. Clin. Microbiol. 21(6); 1985 pp972-979), and it has been suggested that the mannose concentration is useful for diagnosis of these diseases.
As a method for measuring mannose, gas chromatography method (Clin. Chem 25; 1979; pp1384-1387), liquid chromatography method (Bulletin of Japan Urology Association, 80; 1989; pp1816-1823), enzyme method (Clin. Chem 30(2); 1984 pp293-294) and the like are known.
For example, in the enzyme method, since it is hard to conduct accurate quantitative determination if a specimen or sample contains glucose, it is necessary to eliminate the glucose. In the method of Soyama et al (Clin. Chem 30; 1984 pp293-294), glucose in the specimen is preliminarily eliminated by using glucose oxidase and catalase. Then, in the presence of adenosine triphosphate (hereinafter referred to as ATP), mannose is reacted with hexokinase to convert it to mannose 6-phosphate, and then reacted with mannose 6-phosphate isomerase to convert it to fructose 6-phosphate, and further reacted with glucose 6-phosphate isomerase to convert it to glucose 6-phosphate, and at the last, in the presence of oxidative nicotinamide adenine dinucleotide as a coenzyme (hereinafter referred to as NAD), reacted with glucose 6-phosphate dehydrogenase, and then the absorbance (340 nm) of the formed reductive nicotinamide adenine dinucleotide (hereinafter referred to as NADH) is measured by a spectrophotometer to quantitatively determine the mannose.
Further, as an improvement of the above enzyme method, a method for quantitatively determining mannose is reported by Pitkanen et al (Eur. J. Clin. Chem. Clin. Biochem 35(10); 1997 pp761-766), wherein hexokinase and glucose 6-phosphate isomerase are reacted to a specimen wherein glucose and fructose co-exist in the presence of ATP and an oxidative nicotinamide adenine dinucleotide phosphate (hereinafter referred to as NADP), thereby converting the glucose and fructose to glucose 6-phosphate; glucose 6-phosphate dehydrogenase is further added thereto to convert the glucose 6-phosphate to gluconolacton 6-phosphate; the formed reductive nicotinamide adenine dinucleotide phosphate (hereinafter referred to as NADPH) is degraded under acidic condition with hydrochloric acid in order to eliminate the influence of glucose and the like; and then in the same manner as above, in the presence of ATP and NADP, mannose is reacted with hexokinase, mannose 6-phosphate isomerase, glucose 6-phosphate isomerase and glucose 6-phosphate dehydrogenase, and the absorbance (340 nm) of the formed NADPH is measured.
However, in the above-mentioned enzyme methods, since it is required to conduct a complicated enzyme conjugated system wherein various enzymatic reactions are combined, it is difficult to optimize the conditions of all enzymes. Further, since various expensive enzymes are combined for use, there is a problem in the aspect of costs. Furthermore, there is a drawback that these methods are susceptible to influences of components derived from biological specimens or impurities.
On the other hand, in the gas chromatography method and liquid chromatography method, since it is necessary to conduct cumbersome operations such as derivation or labeling of e.g. fluorescence, and to use special apparatuses, these methods are not suitable for treatment of many specimens.
It is an object of the present invention to provide a method for quantitatively determining mannose and a reagent for quantitative determination, by which it becomes possible to conduct treatment of many specimens accurately and conveniently.
In order to accomplish the above object, in one aspect, the present invention provides method for quantitatively determining mannose, which comprises reacting mannose in a specimen with an enzyme which is capable of oxidizing the mannose by dehydrogenation, in the presence of an electron acceptor, and quantitatively determining a formed reductant of the electron acceptor.
In the above method, when the specimen contains glucose in addition to the mannose, it is preferred that, before the reaction with the enzyme, the glucose in the specimen is converted by a glucose eliminator into a structure that is not reactive with the enzyme.
In the above method, the enzyme is preferably a glucose dehydrogenase which belongs to an enzyme number EC class 1.1.1.119, more preferably a aldohexose dehydrogenase which is derived from a microorganism belonging to a gluconobacter genus. As the specimen, it is preferred that the specimen is at least one biological specimen selected from the group consisting of blood, serum, plasma, cerebrospinal fluid and urine, or a specimen prepared from said biological specimen.
In another aspect, the present invention provides a reagent for quantitative determination of mannose, which comprises an enzyme which is capable of oxidizing mannose by dehydrogenation in the presence of an electron acceptor, and an electron acceptor useful for the enzyme.
The reagent preferably further contains a glucose eliminator. Furthermore, the enzyme is preferably a glucose dehydrogenase which belongs to an enzyme number EC class 1.1.1.119, more preferably an aldohexose dehydrogenase which is derived from a microorganism belonging to a gluconobacter genus. Moreover, the glucose eliminator preferably contains a glucose 6-position phosphorylating enzyme, and adenosine triphosphate. Further, the electron acceptor is NADP as a coenzyme.
According to the present invention, since an enzyme which reacts directly with mannose and is capable of oxidizing mannose by dehydrogenation, is used without conducting the measurement through the complicated enzyme conjugated system, it is possible to quantitatively determine mannose conveniently and conduct treatment of many specimens. Further, when the glucose eliminator is used, since the measurement is hardly influenced by the glucose in the specimen, it is possible to quantitatively determine mannose accurately.